DESCRIPTION: The long term objective of this proposal is to understand the mechanism of protein folding, the process by which a newly synthesized polypeptide adopts its biological conformation. A general protein folding intermediate is the molten globule, a species characterized by compactness, near-native levels of secondary structure, and the absence of rigid, specific side chain packing. One of the best studied molten globules is that of alpha-lactalbumin (alpha-LA), a small two-domain protein. The richness of existing knowledge, the ability to probe the backbone topology by disulfide bond formation, and the availability of recombinant variants that are molten globules under non-denaturing conditions make alpha-LA an ideal model system for understanding the function of this intermediate in protein folding. The helical domain of alpha-LA molten globule has a native-like tertiary fold, which serves as a scaffold for organization of the rest of the polypeptide chain and a starting point from which to search for the correct side chain packing. The specific aims of this study are: (1) To understand the molecular interactions and the information in the primary sequence that determine the native-like tertiary fold in the alpha-LA molten globule. (2) To characterize the side chain dynamics in the molten globule and understand how the dynamics change upon formation of the native protein. To achieve these goals: (1) Alanine scanning mutagenesis and pairwise alanine substitutions will be used to determine the contribution of each side chain and its interaction to the specificity for formation of the native-like tertiary fold. (2) NMR relaxation measurements on selectively isotope labeled proteins will be used to study the dynamics of individual residues in the molten globule and to understand the transition from the molten globule to the native state of alpha-LA. These studies will provide foundations for understanding the molecular basis of human diseases caused by protein misfolding or aggregation, as well as for rational design of proteins with specific medical applications.